1. Field of the Invention
The present invention relates to an apparatus and a method in which differences in fluorescence intensity at narrow strips of local portions of individual chromosome or cell flowing through a flow chamber are measured, so that chromosomes or cells of a specific type are sorted and collected on the basis of patterns of the differences.
2. Description of the Prior Art
In order to classify chromosomes or cells of a specific type, various methods have been used in the fields of medical science, biology and the like.
Heretofore, such a method in which chromosomes are classified in accordance with parameters relating to the shape of a chromosome such as a size of the chromosome, e.g., a length of a long-arm portion or a short-arm portion of the chromosome, centrometric index and the like on the basis of the image by an optical microscope has been adopted as a method for classifying chromosomes. However, such a method in which a chromosome in a cell is stained with quinacrine mustard, and a type of chromosomes is identified on the basis of a difference in patterns of fluorescent lateral fringes (Q-band) which indicate characteristic features of the chromosome was discovered by T. Casperson in Sweden in 1970. A method for staining chromosome according to the above described method is called as Q-differential staining. Other than this Q-differential staining, C-differential staining and G-differential staining were discovered and by which a type of chromosome is classified on the basis of a difference in lateral fringes of a higher fluorescence intensity which are called as C-band and G-band differing from Q-band. As described above, at present various chromosome types can be accurately and fully identified on the basis of a difference in fluorescent band patterns appearing on a chromosome.
In order to identify gene of eucaryote, a method for making clear genes of individual chromosomes is employed by the properties of genes contained in the chromosome of a respective type. For this purpose, it is required to collect only a large amount of gene of a specific type in living conditions. Furthermore, in the fields of tumorlogy and immunology, it becomes necessary to collect a large amount of only cells of a specific type such as subgroup of lymphocyte in living conditions.
As a method for classifying cells, there are a method in which morphologic characteristic features of cells or differences in physical properties of cells are skillfully utilized, and a method in which differences in surface membrane of cells are utilized Moreover, such a method in which the surface of a cell is labelled with a specific fluorescent material to identify a variant of the cell by means of fluorescence microscope has been effected.
In addition to the methods as described above, such a manner that a device called as a cell sorter begins to spread with rapidity. In the cell sorter, cells or chromosomes which have been previously labelled with a fluorescent material, flow through a flow chamber or in a jet stream from a flow chamber nozzle at a high-speed. These cells or chromosomes are irradiated with a laser beam, and a fluorescent intensities of the chromosomes or cells as a result of the irradiation is measured, and the measured fluorescent intensities are analyzed, whereby specific chromosomes or cells are collected and sorted. This method is of a special importance in the fields of cytochemistry, immunology, tumorlogy, genetics, molecular biology and the like.
Nevertheless, in a conventional cell sorter, cells or chromosomes have been irradiated with a laser beam having a far larger section than that of a size of the cells or chromosomes flowing through a tube-shaped aqueous solution, so that the intensity of fluorescence emitted from the cells or chromosomes which had been previously labelled as a result of the irradiation has been measured Therefore, a conventional apparatus is called as a zero-resolution apparatus. In these circumstances, the differences in fluorescence intensity derived from local points of cells or chromosomes could not be identified. For this reason, cells or chromosomes of a limited specific type could not be sorted and collected.
For instance, a method which is utilizing a dual beam apparatus has been proposed by P. N. Dean et al. ("High Resolution Dual Laser Flow Cytometry", J. Histochem. Cytochem., Vol. 26, pp. 622-627, 1978.) In this method, LLL 761 chromosomes derived from human pellicle cell are broken up into pieces, then these are stained with fluorescent materials of Hoechst 3325Z and Chromomycin A3, and thus the stained pieces are irradiated with two laser beams of ultraviolet light and visible light, whereby fourteen types of chromosomes are uniquely identified in accordance with such high resolution analysis. However, human ninth to twelfth chromosomes could not be sorted and collected by means of a conventional cell sorter and even the dual beam apparatus.
Then, R. V. Lebo et al., indicated that the human ninth chromosome can be separated from a group of the human tenth to twelveth choromosomes by labelling the human chromosomes with two kinds dyes of DIPI and chromomycin (R. V. Lebo et al., "Science," Vol. 225, 6 Jul., 1984, pp. 57-59). However, the human tenth to twelfth chromosomes can not be separated and collected by means of a conventional cell sorter.
As a method for improving a resolution power of a conventional flow cytometer, it is important how is a laser beam with which chromosomes or cells are to be irradiated formed. L. L. Wheeless et al., and Cambier et al., have developed the apparatuses in which the maximum radius of cell nucleus of a flowing cell and the size of the cell are measured. (L. L. Wheeless: "Slit-Scanning and Pulse Width Analysis", pp. 125-135, in Flow Cytometry and Sorting, John Wiley and Sons, 1979. J. L. Cambier et al.: "A Multidimensional Slit-Scan Flow System", J. Histochem. Cytochem., pp. 321-324, 1979)
However, in the above-mentioned apparatuses, since the slit width is wide, when a chromosome or cell passes through the flow chamber the variation of the size of chromosome or cell with time is not measured every moment, but only the maximum radius of chromosome or cell at the time of passage is measured.
On the other hand, a research group in the Lawrence Livermore National Laboratory has developed a method for narrowing laser beam in which two laser beams are interefered and the obtained fringe-pattern is used (Norgren et al.: "Resoration of Profiles from Slit-Scan Flow Cytometry", IEEE Transactions on Biomedical Engineering, Vol. BME-29, pp. 101-106 (1982)). For example, according to the Young interference method and the Mach-Zehnder interference method, a vertical fringe pattern can be formed. Furthermore, a ring-shaped fringe pattern can be formed in accordance with the Fabry-Perot interference method.
A method in which flowing chromosomes at a high-speed are irradiated with a fringe pattern formed in accordance with the Mach-Zehnder interference method is known by Richard M. Norgren et al., U.S. Pat. No. 4,596,036 "Method and Apparatus for Fringe-Scanning Chromosome Analysis".
In the method of R. M. Norgren et al., however, since flowing chromosomes are irradiated with laser beams of a pattern having several fringes and a high beam intensity, the measurement of such fluorescence intensity emitted by several laser beams of vertical fringes means to measure the total amount of the fluorescence intensity. Accordingly, it is concluded that the fluorescence intensity emitted from the local portions of the vertical fringe of chromosome can not be obtained so far as data as to the total amount of fluorescence intensity are analyzed. For this reason, it was impossible to measure a fluorescent band pattern, a length and the like of chromosome in real-time in accordance with conventional methods.
In this connection, a development for an apparatus and a method for sorting and collecting a specific cell or chromosome while maintaining biological activity on the basis of differences in local fluorescence intensity of chromosome or cell has been strongly desired.